An effective global HIV-1 vaccine is urgently needed. One of the fundamental challenges in HIV-1 vaccine development is the tremendous diversity of HIV-1 strains worldwide. This extreme genetic diversity makes it difficult to design an HIV-1 vaccine that can elicit broadly protective immunity. Vaccines that elicit diverse HIV-1-specific immune responses represent a potential strategy to overcome the challenge of HIV-1 sequence variation. The hypothesis underlying this strategy is that the greater and more diverse the immune responses, the greater the likelihood that there will be a match to the transmitting HIV-1 strain. Currently, we do not know the optimal design of a global HIV-1 vaccine to elicit diverse immune responses. The objective of this K23 Career Development Award is to define HIV-1 vaccine regimens that elicit the greatest diversity of immune responses following immunization. To accomplish this objective, we will use novel cutting-edge immunologic assays to measure how changes in antigen delivery and composition impact the breadth and depth of HIV-1-specific antibodies and T cell responses in humans. Each aim of this K23 award focuses on a different potential vaccine strategy for increasing immune diversity, including a vaccine that combines viral vectors and proteins for HIV-1 antigen delivery (Aim 1), novel mosaic HIV-1 antigens bioinformatically engineered to optimize coverage of global HIV-1 sequences (Aim 2), and a new replicating viral vector for expressing HIV-1 antigens (Aim 3). The Specific Aims are as follows: Specific Aim 1: To evaluate whether immunization with vectored HIV-1 Env antigens (ALVAC-HIV) plus Env protein (AIDSVAX B/E) elicits greater diversity of Env-specific antibodies than immunization with either vectored Env or Env protein alone in humans. Specific Aim 2: To assess if mosaic HIV-1 antigens (MVA-Mosaic) elicit greater diversity of Env-specific antibodies and Gag-specific CD8+ T cells than multi-clade HIV-1 antigens (MVA-Natural) in humans. Specific Aim 3: To evaluate the diversity of serum and mucosal Env-specific antibodies in humans vaccinated with an oral, replicating Ad26 vector expressing mosaic HIV-1 Env (rcAd26.Mos1ENV). The findings from this K23 award have the potential to greatly inform HIV-1 vaccine design. Through the above Specific Aims, we will identify which HIV-1 vaccines elicit the greatest diversity of HIV-1-specific immune responses, and will be able to craft novel HIV-1 vaccine regimens that include combinations of our most optimal candidates. In this way, we aim to advance the development of an effective global HIV-1 vaccine.